The present invention relates to a method for detecting and for quantifying, in diverse biological media, adenovirus nucleic acids by real-time polymerase chain reaction (PCR) measurement. A subject of the invention is also oligonucleotides for carrying out this method.
Gene therapy is currently undergoing considerable development. Adenoviruses, which are naturally responsible for generally benign infections, are among the vectors used because of their many advantages.
The extension of the clinical development of these adenoviral vectors to late phases II and III for the purposes of registration and of authorization for marketing makes it necessary to develop, validate and implement specific and sensitive techniques which allow the measurement of the quantitative and qualitative biodistribution of adenovectors in diverse biological compartments, and the detection of the emergence, at low viral load, of infection with contaminants from the production of adenovectors (in particular replication-competent recombinant adenovectors). It is also necessary to detect intercurrent adenoviral infections which are due to wild-type viruses of various serotypes, which are responsible for the vast majority of the incidence of these natural infections, whether these infections are clinically florid or latent, and to determine the virological kinetics of these wild-type infections and the clinical significance in diverse situations, in the general population or in patients who are at risk (patients suffering from cancer, transplant patients, immunosuppressed or immunodepressed patients, etc.).
The presence of an adenovirus in permissive cell culture is visualized, in the detection techniques routinely used to date, by a cytopathic effect in the cell layer. In order to demonstrate that this cytopathic effect is indeed due to an adenovirus, an antigen which is specific for the adenovirus is detected by the ELISA method.
Techniques for amplifying extracted nucleic acids of adenoviruses by PCR have also been described. Thus, Japanese patent application JP 07327700 describes a PCR-type method in which portions of the DNA encoding hexon, which is an adenovirus capsid protein, are amplified. The amplification products are xe2x80x9cdot-blottedxe2x80x9d, then hybridized with probes which are specific for each serotype. This method is thus intended to identify the subgroups and serotypes, and not to detect and quantify all of the adenoviruses present in a sample. In addition, the sensitivity of this method is low and thus generally unsuitable for precise measurements, such as those required during the monitoring of patients treated by gene therapy.
Pring-Akerblom et al. (Journal of Medical Virology, 58:87-92, (1999), have also described a method which uses six pairs of primers in the same PCR reaction (so-called multiplex PCR). The identification of the subgroup is carried out by loading the PCR products onto electrophoresis gel and then determining the size of the PCR products.
Crawford-Miksza et al. (Journal of Clinical Microbiology, 37:1107-1112, (1999), have described a hexon PCR which specifically detects type 4, 7 and 21 adenoviruses using consensus primers comprising inosine as the nucleic base in the event of mismatching in certain variable position. In that document, nine distinct primers are described.
These prior techniques use a standard PCR and a conventional analysis method which is generally on electrophoresis gel with or without hybridization.
None of these techniques make it possible both to broadly detect adenoviruses originating from the majority of the known serotypes, and to quantify the viral loads thereof with a sensitivity which is suitable for the needs of gene therapy in particular.
The aim of the present invention is thus to resolve these problems by providing a technique which is sensitive and which makes it possible to detect the majority of the adenovirus serotypes.
The applicants have surprisingly shown that such advantages are obtained by amplifying in real time a given sequence of the DNA encoding hexon with the aid of primers having limited degeneracy, and revealing the amplification product with the aid of a nondegenerate probe.
The present invention first of all relates to a method for detecting and/or for quantifying adenovirus nucleic acids in a biological sample wherein:
an adenovirus nucleotide sequence is amplified by real-time PCR using degenerate sense primers and degenerate antisense primers, wherein said primers are chosen from oligonucleotides having at least 80% homology with a sequence between nucleotides 21000 and 22000 of type 5 adenovirus sequence, and which corresponds to sequence SEQ ID No. 4, and oligonucleotides comprising a complementary sequence thereof, and
an amplification reaction product may be detected using a nondegenerate probe comprising an oligonucleotide having at least 80% homology with a sequence between nucleotides 21000 and 22000 of type 5 adenovirus sequence, which corresponds to sequence SEQ ID No. 4, and oligonucleotides comprising a complementary sequence thereof, wherein said product may be detected during a number of amplification reaction cycles which is sufficient to allow the production of a measurable amount of amplification product.
The term xe2x80x9creal-time PCRxe2x80x9d is intended to mean any amplification technique which makes it possible to monitor the evolution of an ongoing amplification reaction. Polymerase Chain Reaction is abbreviated as xe2x80x9cPCRxe2x80x9d.
Generally, sequences of the primers and/or of the probe may comprise at least 80% homology with at least one sequence between nucleotides 540 and 780 of sequence SEQ ID No. 4, or with a complementary sequence thereof. The two primers are chosen on the sense and antisense strands, respectively, and in such a way as to allow the amplification of a DNA fragment. With regard to the probe, it is chosen so as to hybridize with the DNA fragment resulting from the amplification. The PCR primers in accordance with the present invention which are used to amplify the target adenovirus nucleic acid in a sample are located in a region which is constant for the hexon gene of human adenoviruses, and the hybridization site of the probe in accordance with the present invention is located between the two primers. In one embodiment, the adenoviruses are of human tropism.
Generally, said oligonucleotides may comprise at least 15 nucleotides, and the probe has a theoretical melting temperature Tm which is higher than the theoretical Tm of the primers by approximately 10xc2x0 C.xc2x10.5.
A method of the invention is termed hexon PCR method in abbreviated form in the remainder of the text. It comprises a repetition of a cycle comprising:
separation of the strands to be amplified by heating the DNA extracted from the sample,
hybridization of the probe,
hybridization with primers as defined above, and
elongation with a polymerase.
In one embodiment, the amplification method comprises the sense and antisense primers HEX1 and HEX2 described below. In a further embodiment, the amplification product is hybridized with the HEX probe described below.
One subject of the present invention is an oligonucleotide, comprising at least 10 consecutive nucleotides of the following sequence SEQ ID No. 1 or of a sequence having at least 80% sequence homology with said sequence:
5xe2x80x2-YCC CAT GGA YGA GCC CAC MCT-3xe2x80x2
in which Y represents C or T, and M represents A or C.
In one embodiment of the invention, said oligonucleotide has at least 90% sequence homology with said sequence. In another embodiment, said oligonucleotide has at least 95% sequence homology with said sequence.
In yet another embodiment, said oligonucleotide comprises between 15 and 30 nucleotides.
One subject of the present invention is a HEX1 sense primer comprising a mixture of oligonucleotides which satisfy this definition. The HEX1 primer has three degeneracies at positions 1, 10 and 19 which make it possible to cover the majority of the serotypes.
Another subject of the present invention is a second oligonucleotide, comprising at least 10 consecutive nucleotides of the following sequence SEQ ID No. 2 or of a sequence which shows at least 80% sequence homology with said sequence:
5xe2x80x2-GAG AAS GGB GTG CGC AGG TAS-3xe2x80x2
in which S represents G or C, and B represents C, G or T.
In one embodiment of the invention, said second oligonucleotide has at least 90% sequence homology with said sequence. In another embodiment, said second oligonucleotide has at least 95% sequence homology with said sequence.
In yet another embodiment, said second oligonucleotide comprises between 15 and 30 nucleotides.
Another subject of the present invention is a HEX2 antisense primer comprising a mixture of oligonucleotides which satisfy this definition. The HEX2 primer has three degeneracies at positions 6, 9 and 21 which make it possible to cover the majority of the serotypes sequenced.
In one embodiment of the invention said primers comprise at least two oligonucleotides. In other embodiments, said primers may comprise three or four oligonucleotides.
The positions of the sequences SEQ ID No. 1 and 2, which are located in the 3xe2x80x2 portion of the Open Reading Frame (xe2x80x9cORFxe2x80x9d) encoding the Hexon protein, are, talking the complete sequence of the type 5 adenovirus (filing No. M73260) as reference, 21565 (HEX1) and 21656 (HEX2), respectively. They correspond to positions 21048 and 21139 of the Ad5CMVp53 sequence (length 35308 bp).
The applicants have shown that primer concentration is a parameter of real-time PCR. Therefore, the degeneracy of the HEX1 and HEX2 primers was limited.
A subject of the present invention is also a third oligonucleotide, comprising at least 10 consecutive nucleotides of the following sequence SEQ ID No. 3 or of a sequence having at least 80% sequence homology with said sequence, or with a sequence complementary to said sequence:
5xe2x80x2-CAC CAG CCA CAC CGC GGC GTC ATC GA-3xe2x80x2. 
In one embodiment of the invention, said third oligonucleotide has at least 90% sequence homology with said sequence. In another embodiment, said third oligonucleotide has at least 95% sequence homology with said sequence.
In yet another embodiment, said third oligonucleotide comprises between 20 and 35 nucleotides.
A subject of the present invention is also a HEX probe comprising an oligonucleotide having this sequence.
It also relates to oligonucleotides having a sequence complementary to those of the oligonucleotides mentioned above.
In one embodiment of the present invention, the probe comprises a revealing molecule or revealing system of molecules. Said revealing system may comprise a reporter dye and a quenching dye, which are bound to the 5xe2x80x2 and 3xe2x80x2 ends, respectively, of the probe. In one embodiment, said reporter dye is a fluorescent dye and said quenching dye is a fluorescence-quenching dye. According to another embodiment, the revealing system consists of a reporter/quencher pair 6-carboxyfluorescein (FAM) and
6-carboxytetramethyl-rhodamine (TAMRA) bound in the 5xe2x80x2 and 3xe2x80x2 positions, respectively, of the probe. In yet another embodiment, one of the primers is labeled in this way with a reporter/quencher pair and said primer then performs the role of a probe.
In one embodiment of the invention, the probe is:
5xe2x80x2FAM-CAC CAG CCA CAC CGA GGC GTC ATC GA-TAMRA 3xe2x80x2
Said revealing system can also optionally be a so-called tailing revealing system. Wherein said tailing revealing system comprises at one of the ends of the probe, a tail which can self-pair. This self-pairing is detected with the aid of a label which binds specifically to the sequence in this configuration. In the presence of a target, unpairing takes place and a signal is given off. In the absence of a target, the signal is not given off. Other revealing systems can also be used. In another revealing system, detection may involve hybridization of probe to said amplification product and a signal is given off.
As a variation, the sense and antisense primers can comprise a revealing molecule or revealing system of molecules.
The PCR product obtained by the method which is the subject of the present invention, using the primers of sequences SEQ ID No. 1 and SEQ ID No. 2, may generally be 114 bp and may have a GC% of approximately 58.8%.
The polymerase used in the present invention may be any enzyme which has polymerase activity and which can be used under the PCR operating conditions. In one embodiment, the polymerase is Taq polymerase.
A subject of the present invention is also a kit of reagents for real-time PCR-type amplification reaction for detecting adenoviruses, comprising a pair of sense and antisense primers and a probe as described above. Said kit may also comprise negative controls and/or positive controls, such as, for example, two negative controls and two positive controls. Said kit may comprise at least one separate compartment, wherein each compartment may comprise at least one of said primers or probe. Said kit may comprise two or more compartments.
A subject of the present invention is also a PCR method for detecting and/or quantifying adenoviruses in a sample to be measured which is likely to contain them, comprising a real-time PCR which uses a kit according to the invention.
According to one embodiment of the invention, said two positive controls are extracted in parallel to a series of samples to be assayed, and comprise a standard comprising a purified and titered adenovirus solution and a calibrator which is suitable for the type of sample to be measured.
The method in accordance with the invention may be applicable to detecting and/or quantifying adenoviruses present in samples such as culture suspension or supernatant, plasma, urine, oropharyngeal washes, lymphocytes, seminal fluids, tumor or nontumor biopsies, rectal swabs, feces or ascites.
The present invention also relates to a method for selecting adenoviruses which are useful as vector candidates by evaluating the replication performances of the adenoviruses using a detection and/or quantification method according to the invention to monitor adenoviral multiplication kinetics.
The present invention also relates to a method for diagnosing the serotype of adenoviruses present in a sample to be tested, comprising implementing a method of the invention, and then in sequencing the PCR product obtained.
The nucleotide sequences of the various serotypes encoding the Hexon protein were obtained from publicly-available databanks, or were sequenced by conventional methods. The Ad3, Ad7, Ad12, Ad14, Ad16 and Ad21 serotypes have an A residue at position 9 of the HEX probe. The Ad5, Ad1, Ad2, Ad6, Ad13, Ad40 and Ad48 serotypes have a G residue at this same position, whereas the Ad4 and Ad41 serotypes have a C residue. The sequence which has an A residue was chosen, since the G-type sequence, unlike the A-type or C-type sequences, has a very highly stable secondary structure (DG=xe2x88x927.1 kcal/mol) which is likely to disturb its hybridization and/or its displacement and/or its degradation during polymerization. On the other hand, the A-type sequence is more common than the C-type sequence.
The A/C (Ad5CMVp53 and 5,1, 2, 6, 13, 40 and 48 serotypes) or A/G (4 and 41 serotypes) mismatches cause a decrease in the delta Rn without significant modification of the Ct (delta Rn is the difference in fluorescence detected between the measured fluorescence of the background noise and the detected fluorescence of the sample to be tested; Ct is defined as the number of fractional cycles in which the fluorescence generated by cleaving the probe significantly exceeds, in general by 10-fold, the background noise).
Ad5, Ad4, Ad12 and Ad14 serotypes and Ad5CMVp53 have additional differences at position
5 for the Ad5 serotype and Ad5CMVp53
24 for the Ad4 and Ad12 serotypes
3, 12 and 19 for the Ad14 serotype
Despite these differences, it is possible, surprisingly, to detect the nucleotide sequences encoding the hexon proteins of these serotypes.
One of the advantages of the present method for detecting and/or for quantifying human adenoviruses, with respect to the methods described in the prior art, is an outstanding detection sensitivity with a detection threshold of 10 particles or 1 pfu (plaque forming unit) by PCR reaction.
Another advantage lies in the universality of the method, since it has been possible to detect and quantify 17 different serotypes of human adenoviruses, which are representative of all of the subgroups A to F.
In addition, the analysis of the PCR products is carried out directly at the end of the PCR cycles by reading the fluorescence obtained during the cycles. It is not therefore necessary to work with PCR products which are at risk of being contaminated for the subsequent analyses.
Furthermore, the raw data may be completely recorded and preserved. Specifically, each PCR analysis may be stored as computer files which can be archived for a long period of time without alteration. The raw data can be re-analyzed at any moment, permitting re-analysis if criteria change. Conserving of the raw data is essential when it is necessary to meet the standards of Good Laboratory Practice.
In addition, the quantification of the number of targets placed in the reaction at the start is generally very reliable and reproducible. The detection of the PCR product is carried out during the PCR cycles, often with the aid of a fluorescent probe or other reporter system. This is necessary to detect the PCR product, and generally takes place in the middle of the exponential phase of PCR, and not at the end point; this detection principle is thus more sensitive and more specific.
Another advantage lies in the fact that nonspecific amplifications are avoided due to the hot start principle, the real-time PCR being carried out in the presence of a heat-stable DNA polymerase which is activated at the first denaturation.
In implementing the present invention, reference may optionally be made to a general review of PCR techniques, and to the explanatory note entitled xe2x80x9cQuantitation of DNA/RNA Using Real-Time PCR Detectionxe2x80x9d published by Perkin Elmer Applied Biosystems (1999) and to PCR Protocols (Academic Press New York, 1989).